Rapid fluorescence visualization of Yersinia enterocolitica by CRISPR/Cas12a using novel specific target obtained by pan-genome analysis

Abstract

Molecular detection methods are often used to detect the presence of common pathogenic bacteria. However, molecular detection methods for Yersinia enterocolitica (Y. enterocolitica), an important foodborne pathogen, are rarely reported. This is attributable to the need for better detection targets. Therefore, this study aims to obtain a species-specific target for detecting Y. enterocolitica through pan-genome analysis using public genome resources. The YE5303_14291 gene was identified as a candidate target, and a YE5303_14291-based CRISPR/Cas12a fluorescence visualization and test strip platform were developed. The developed PCR-CRISPR/Cas12a and RAA-CRISPR/Cas12a systems can quantitatively detect Y. enterocolitica, with linear ranges from 3.1 × 1010 CFU mL−1 to 3.1 × 10° CFU mL−1, and the limit of detection (LOD) was 3.1 × 10° CFU mL−1. The detection systems all showed reasonable specificity, and the actual sample detection results were consistent with the isolation and culture results. In conclusion, the CRISPR/Cas12a detection system based on the target YE5303_14291 has the potential to specifically and rapidly detect Y. enterocolitica in food samples.